Voltage sensitive phosphatases: emerging kinship to protein tyrosine phosphatases from structure-function research

The transmembrane protein Ci-VSP from the ascidian Ciona intestinalis was described as first member of a fascinating family of enzymes, the voltage sensitive phosphatases (VSPs). Ci-VSP and its voltage-activated homologs from other species are stimulated by positive membrane potentials and dephosphorylate the head groups of negatively charged phosphoinositide phosphates (PIPs). In doing so, VSPs act as control centers at the cytosolic membrane surface, because they intervene in signaling cascades that are mediated by PIP lipids. The characteristic motif CX5RT/S in the active site classifies VSPs as members of the huge family of cysteine-based protein tyrosine phosphatases (PTPs). Although PTPs have already been well-characterized regarding both, structure and function, their relationship to VSPs has drawn only limited attention so far. Therefore, the intention of this review is to give a short overview about the extensive knowledge about PTPs in relation to the facts known about VSPs. Here, we concentrate on the structural features of the catalytic domain which are similar between both classes of phosphatases and their consequences for the enzymatic function. By discussing results obtained from crystal structures, molecular dynamics simulations, and mutagenesis studies, a possible mechanism for the catalytic cycle of VSPs is presented based on that one proposed for PTPs. In this way, we want to link the knowledge about the catalytic activity of VSPs and PTPs.DFG, 53182490, EXC 314: Unifying Concepts in CatalysisDFG, 390540038, EXC 2008: UniSysCa

Investigation of the intramoleculare coupling process of the voltage sensitive phosphatase Ci-VSP

Phosphatidylinositol-Phosphate (PIP) sind an einer Vielzahl intrazellulärer Signalkaskaden beteiligt. Funktionseinschränkungen von Proteinen, die in den PIP-Metabolismus eingreifen, führen zu schweren Krankheiten, wie z.B. Krebs. Die spannungssensitive Phosphatase Ci-VSP aus der Seescheide Ciona intestinalis ist ein Modellprotein, an dem PIP-selektive Katalysemechanismen auf molekularer Ebene untersucht werden. Ci-VSP besteht aus zwei funktionellen Modulen: (1.) einer transmembranen Spannungssensor- (VSD) und (2.) einer zytosolischen katalytischen Domäne (CD), die nochmals in eine Phosphatase- (PD) und eine C2-Domäne unterteilt wird. Beide Module sind über die mehrfach positiv geladene Linkersequenz M240-K257 miteinander verbunden. Bei depolarisierenden Membranpotentialen laufen in der VSD Konformationsänderungen ab, die zur Aktivierung der PD führen, wodurch sie selektiv die Kopfgruppen von PIP-Membranlipiden dephosphoryliert. Im VSD-CD-Kopplungsprozess spielt der Linker eine essentielle Rolle, da er nach der Aktivierung der VSD über elektrostatische Wechselwirkungen an die negativ geladene Membranoberfläche bindet und dabei die CD an die Membran rekrutiert, wodurch die enzymatische Katalyse initiiert wird. Um die funktionelle Rolle des Linkers detaillierter aufzuklären, wurde im Rahmen dieser Arbeit eine Cystein-Scanning-Mutagenese der gesamten M240-K257-Sequenz durchgeführt. Die generierten Mutanten wurden in den Oozyten des Krallenfroschs Xenopus laevis heterolog exprimiert und elektrophysiologisch mittels der Zwei-Elektroden-Spannungsklemme charakterisiert. Die Analyse der PD-Aktivität erfolgte in Koexpressionsexperimenten mit den PI(4,5)P2-sensitiven Kaliumkanälen KCNQ2/KCNQ3. Zudem wurde die VSD-Dynamik aller Ci-VSP-Mutanten untersucht. Hierbei wurde festgestellt, dass sich N-terminale Linkermutationen (M240C-K257C) weniger dramatisch auf die VSD-CD-Interaktion auswirken als C-terminale (Q250C-K257C). Mit A242C, R245C, K252C und Y255C wurden vier Konstrukte mit einer vollständig aufgehobenen PD-Aktivität identifiziert. Aufgrund der Periodizität des Mutationseffekts im N- und C-terminalen Linker wurden alpha-helikale Strukturelemente in diesen Bereichen vermutet. Außer K252C wiesen alle katalytisch inaktiven Mutanten zudem eine beschleunigtere VSD-Kinetik auf als das unmutierte Protein (Wildtyp). Durch die K257Stop-Mutante, bei der die CD des Proteins deletiert war, konnte nachgewiesen werden, dass der Verlust der CD-Membranbindung die Beschleunigung der VSD-Kinetik hervorrief. Hieraus wurde geschlussfolgert, dass die Mutationen A242C, R245C und Y255C die Proteinmodule voneinander entkoppeln. Im Gegensatz dazu wies die katalytisch inaktive Mutante K252C eine dem Wildtyp ähnliche VSD-Kinetik auf und schien somit nicht für die Membranbindung der CD verantwortlich zu sein. Daher wurde vermutet, dass der Linker noch andere Prozesse als nur die Membranbindung der CD vermitteln könnte. Um diese Frage zu klären, wurde ein dreidimensionales Homologiemodell der Ci-VSP-Struktur konstruiert, dessen Dynamik simuliert wurde. Anhand der Simulationsergebnisse konnte bekräftigt werden, dass sich im gesamten, überwiegend alpha-helikal strukturierten Linker Aminosäuren befinden, die über Salzbrücken mit den negativ geladenen Lipidkopfgruppen an der Membran interagieren. Hierbei spielt R245 eine besondere Rolle, da die Interaktionen dieser Seitenkette mit der Membran einen strukturellen Bruch im Linker verursachen, wodurch sich der C-terminale Linkerbereich zur PD hin ausrichtet und sich Salzbrücken zwischen K252, R253 im Linker und D400 und E402 in der PD ausbilden. D400 und E402 befinden sich dabei in einem von drei Loopregionen, die die katalytische Bindungstasche der PD bilden und stabilisieren, dem TI-Loop. Von den Wechselwirkungen zwischen Linker und TI-Loop wurde die R253-D400-Salzbrücke als die kritischste für die Ci-VSP-Funktionalität identifiziert, da Mutationen beider Positionen zu einer Modulentkopplung führten, was sich in einer nahezu aufgehobenen PD-Aktivität und einer beschleunigten VSD-Kinetik widerspiegelte. Weitere Untersuchungen zeigten auf, dass der Kontakt zwischen Linker und TI-Loop neben den elektrostatischen auch durch hydrophobe Interaktionen stabilisiert wird, die insbesondere durch die Aminosäuren Y255 im Linker und F401 im TI-Loop vermittelt werden. Zudem stabilisieren auch die negativ geladenen Kopfgruppen an der Membranoberfläche die Interaktion zwischen Linker und TI-Loop. Anhand der hier erhaltenen Daten wurde eine Hypothese über den Ablauf des linkervermittelten VSD-CD-Kopplungsprozesses entwickelt, bei dem die Interaktionen zwischen Linker und TI-Loop entscheidend für die Formierung der katalytischen Bindungstasche und somit für die Aufrechterhaltung der enzymatischen Aktivität der PD ist. Mit diesen Ergebnissen wird das Wissen über den spannungsabhängigen Aktivierungsmechanismus des Ci-VSP-Proteins erweitert und vertieft.Phosphatidylinositol phosphates (PIP) are involved in a variety of intracellular signaling cascades. Proteins intervening in these pathways are of huge interest in medical research, because their insufficient functionality is associated with the development of serious diseases, e.g. cancer. The voltage sensitive phosphatase Ci-VSP from the ascidian Ciona intestinalis is one model system to investigate PIP-selective catalysis on a molecular level. Ci-VSP consists of two functional modules: (1.) a transmembrane voltage sensor domain (VSD) and (2.) a cytosolic catalytic domain (CD), which comprises a phosphatase (PD) and a C2-domain. Both modules are connected via the highly positively charged linker sequence M240-K257. Upon depolarization of the plasma membrane, conformational changes of the VSD lead to the activation of the PD, which selectively dephosphorylates PIP-lipids at the membrane surface. For the coupling process between VSD and CD, the linker seems to play a crucial role, because it could interact electrostatically with the negatively charged membrane surface during the activation of the VSD, which results in the recruitment of the CD to the membrane and therefore in the initiation of enzymatic catalysis. To investigate the role of the linker for the intermodular coupling process in more detail, a cysteine scanning mutagenesis of the whole sequence M240-K257 was carried out in this work. The different Ci-VSP mutants were heterologously expressed in the oocytes of the claw frog Xenopus laevis and functionally characterized by the two-electrode voltage-clamp technique. The PD activity was analyzed in coexpression experiments with the PI(4,5)P2-sensitive potassium channels KCNQ2/KCNQ3. Additionally, the mutation effect on the VSD dynamics was studied for all Ci-VSP mutants. In this work, it can be demonstrated that mutations in the N-terminal linker (M240C-S249C) are less destructive for the module interaction in Ci-VSP than mutations in the C-terminal sequence (Q250C-K257C). With A242C, R245C, K252C, and Y255C, four linker mutants were identified with a completely abolished PD activity. Because the mutation effect occurred periodically, alpha-helical elements in the linker region were proposed. Except for K252C, all inactive mutants showed faster VSD kinetics than the unmutated protein (wild type). The results of the K257Stop-mutant, in which the cytosolic domain was deleted, clarified that an increase in VSD flexibility is due to a lack of membrane binding of the CD. Therefore, it was concluded that the mutations A242C, R245C, and Y255C lead to an uncoupling of the VSD and CD. On the contrary, the inactive K252C-mutant showed almost identical VSD kinetics to the wild type indicating that K252 is not critical for the membrane binding of the CD. Therefore, it was presumed that this position is involved in other processes than electrostatic interactions with the membrane to enable the enzymatic catalysis. To shed more light into this question, a three-dimensional homology model of Ci-VSP was constructed, whose structural dynamics was predicted by molecular dynamics simulations. With the simulation results, it could be confirmed that several amino acids of a mainly alpha-helical structured linker interact electrostatically with the negatively charged membrane surface. The position R245 was identified to play a special role in this interaction, since the formation of salt bridges between R245 and phosphate groups of PIP at the membrane led to a structural break in the M240-K257-motif and therefore to a spatial approach of the C-terminal linker to the PD. In this way, stable salt bridges were formed between K252 and R253 in the linker and D400 and E402 in the PD. Interestingly, the positions D400 and E402 are localized in the TI loop, which is one of three loop regions that form the substrate binding pocket of the PD. From all interactions, the salt bridge between R253 and D400 seems to be the most crucial one for the VSD-CD interplay, because mutations of both positions led to a uncoupling of the Ci-VSP modules which is mirrored in a strongly reduced PD activity and fast VSD kinetics. In addition to this salt bridge, the connection between linker and TI loop is further stabilized by hydrophobic interactions that involve the residues Y255 in the linker and F401 in the PD. Moreover, the negatively charged head groups of membrane lipids further stabilize the interactions in the linker-TI loop interface. Based on these results, a hypothesis about the linker-mediated coupling process was developed, in which the formation of correct interactions between linker and TI loop is the essential step for those conformational changes in the PD that are required for initiating the enzymatic catalysis. In summary, the findings of this work broaden and deepen the knowledge about the voltage-dependent activation process in the Ci-VSP protein

The Linker Pivot in Ci-VSP: The Key to Unlock Catalysis

<div><p>In the voltage-sensitive phosphatase Ci-VSP, conformational changes in the transmembrane voltage sensor domain (VSD) are transduced to the intracellular catalytic domain (CD) leading to its dephosphorylation activity against membrane-embedded phosphoinositides. The linker between both domains is proposed to be crucial for the VSD-CD coupling. With a combined approach of electrophysiological measurements on <i>Xenopus</i> oocytes and molecular dynamics simulations of a Ci-VSP model embedded in a lipid bilayer, we analyzed how conformational changes in the linker mediate the interaction between the CD and the activated VSD. In this way, we identified specific residues in the linker that interact with well-defined amino acids in one of the three loops forming the active site of the protein, named TI loop. With our results, we shed light into the early steps of the coupling process between the VSD and the CD, which are based on fine-tuned electrostatic and hydrophobic interactions between the linker, the membrane and the CD.</p></div

Mutations in the TI-loop affect the number of contacts with the linker during the MD simulation.

<p>Interactions between the complete linker, its N- and C-terminal parts (240–249 and 250–257, respectively), K252, R253 and Y255 with the TI loop are summarized in terms of average contacts during the last 30 ns of simulation. Contacts are defined as atoms within a sphere of 3.5 Å.</p

Topology scheme of Ci-VSP.

<p><i>(</i><b><i>A</i></b><i>)</i> Topology scheme of Ci-VSP with the transmembrane voltage sensor (VSD) and the intracellular catalytic domain (CD), which comprises the phosphatase (PD) and the C2-domain. VSD and CD are connected via the linker sequence M240–K257. <i>(</i><b><i>B</i></b><i>)</i> Three-dimensional structure of the Ci-VSP CD based on the crystal structure by Liu et al. (PDB entry 3V0H) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0070272#pone.0070272-Liu1" target="_blank">[14]</a>. The P- (orange), the TI- (blue), and the WPD loop (yellow) form the active site of the CD. The linker (colored in red, with R253 shown as sticks) is oriented toward the TI loop, as proposed by Liu et al. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0070272#pone.0070272-Liu1" target="_blank">[14]</a>. <i>(</i><b><i>C</i></b><i>)</i> Amino acid alignment of Ci-VSP’s linker (M240–K257), active site (H362–R369), and the TI loop (R398–S414) with regions of the homolog PTEN (Hs, Homo sapiens). Amino acid identities are highlighted with a gray background. Putative interacting partners in the linker and the TI loop are denoted with blue and red asterisks, respectively. Single and multiple altered Ci-VSP mutants are aligned as well (in red letters: mutated positions). In the single neutralization mutants, the X stands for N (in case aspartate was the native residue) or for Q (in case of glutamate).</p

Models of WT and mutants reveal differences in the interface between linker and TI loop.

<p>Representative snapshots showing the linker-TI loop interface of <i>(</i><b><i>A</i></b><i>)</i> the WT, <i>(</i><b><i>B</i></b><i>)</i> NEUT, <i>(</i><b><i>C</i></b><i>)</i> ALA, and <i>(</i><b><i>D</i></b><i>)</i> D400A model. For clarity, only K252, R253, Y255, D400X, E402X (X denotes the inserted mutation residue, Fig. 1C) and F401 are shown as indicated in ball-and-stick representation. Furthermore, the backbones of the linker and the TI loop are represented as blue and beige cartoon, respectively. For a better orientation, the membrane lipids are shown as cyan sticks. For the NEUT model, PI(4,5)P₂ molecules interacting with R253 are indicated, with C atoms in black.</p

Phosphatase activities of TI loop mutants.

<p><i>(</i><b><i>A</i></b><i>)</i> Representative currents traces of Ci-VSP WT and denoted mutants that were co-expressed with KCNQ2/KCNQ3 potassium channels in <i>Xenopus</i> oocytes. Channel currents were recorded in response to a depolarization pulse from a holding potential of −80 to+80 mV for the indicated time interval. <i>(</i><b><i>B</i></b><b>–</b><b><i>C</i></b><i>)</i> From the resulting current traces, maximal and minimal current amplitudes (I_max and I_min) were determined during the depolarization phase at+80 mV. Inhibition ratios calculated with the values of I_min and I_max are plotted with the corresponding time durations required for each mutant to inhibit the channel currents to 50% (τ_50%). Inhibition ratio of 0.5 (corresponding to 50%) is highlighted as dashed line.</p

Flipping mechanism of R253 between D400 and E402 in the WT model.

<p>Interaction interface between the linker (blue backbone) and the TI loop (beige backbone) in the WT model. Membrane lipids are represented as cyan-colored sticks. A typical snapshot of the most frequently occurring state is shown in <i>(</i><b><i>A</i></b><i>)</i>. Here, salt bridges between R253 and D400 strongly stabilize the contact between linker and TI loop. In this state, K252 is able to interact with E402. <i>(</i><b><i>B</i></b><i>)</i> The stable configuration between R253 and D400 breaks several times during the 50 ns long MD simulation whereby R253 gets in contact with E402. In this constellation of salt bridges, the interaction between K252 and the TI loop is interrupted.</p

TI loop mutations differently affect the voltage sensor dynamics.

<p>On-sensing currents of Ci-VSP were recorded from a holding potential of −60 mV in response to variable test pulses between −40 to+160 mV at maximum (increment: 20 mV, duration: 500 ms). Off-sensing currents were monitored by stepping back to −50 mV after the test potential phase (off-pulse duration: 500 ms). <i>(</i><b><i>A–D</i></b><i>)</i> Representative full current traces are shown for the WT and the denoted mutants. From these signals, selected off-currents are zoomed out (40 mV increments). As described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0070272#s4" target="_blank">Materials and Methods</a>, Q_off,all-V-distributions were calculated by integrating the off-sensing currents. These distributions are plotted against the potential of the preceding test pulse phase. From the whole amount of translocated sensing charges (Q_off,all), the fast (Q_off,fast) and the slow (Q_off,slow) component was determined as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0070272#s4" target="_blank">Materials and Methods</a>. All calculated Q_off-values were normalized to the Q_off,all-value corresponding to the test potential of+120 mV. Q_off,all-V-distributions were approximated with a Boltzmann-type function (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0070272#s4" target="_blank">Materials and Methods</a>). Parameters of voltage-dependence (V_0.5, z_q) are given in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0070272#pone.0070272.s008" target="_blank">Table S3</a>.</p